ORGANIZATION AND ALTERNATE SPLICING OF THE MURINE FOLYLPOLYGLUTAMATE SYNTHETASE GENE - DIFFERENT SPLICE VARIANTS IN L1210 CELLS ENCODE MITOCHONDRIAL OR CYTOSOLIC FORMS OF THE ENZYME

The organization of the murine folylpolyglutamate synthetase (FPGS) ge ne has been determined by sequence analysis that also revealed an inte resting complexity in the case of exon 1, The entire nucleotide sequen ce of the L1210 FPGS cDNA, the 3'- and 5'-untranslated regions, the mi tochondrial leader sequence, and the coding region were found to be di stributed on 15 exons with an overall length of 10.358 kilobases, Two splice variants of exon 1 were identified by screening of an L1210 cel l cDNA library, Variant I (exons 1a + 1b plus 2-15) incorporates all o f the sequence homologous to the recently reported (Taylor, S. M., Fre emantle, S. J., and Moran, R. G. (1995) Cancer Res. 55, 6030-6034) hum an exon 1, including two ATG start codons at positions +1 and +126, an d encodes both mitochondrial and cytosolic form of FPGS. The most prev alent variant, Variant II (exons Ib plus 2 to 15), incorporates only a portion (92 nucleotides at the 3' end) of this sequence, incorporates only one ATG start codon at position +126, and encodes only cytosolic FPGS, The existence of this variant is consistent with the identifica tion of an appropriately situated internal donor/acceptor site in what was believed to be exon 1, A third related variant (Variant III) with a novel 5' termini was originally identified by screening of a mouse liver cDNA library, This variant, which occurs at moderately low frequ ency in the L1210 cell cDNA library, incorporates an alternate to exon 1a (exon 1c) spliced to exon Ib plus exons 2-15 and encodes a differe nt mitochondrial leader peptide than Variant I. The identification of these variants suggests another possible mechanism, i.e. at the level. of precursor mRNA splicing, for regulating synthesis of mitochondrial versus cytosolic forms of FPGS in the cell, Exon 1c is positioned in the gene upstream of exon 1a separated by an intron of 56 nucleotides within a region of DNA sequence that like the homologous human sequenc e is distinctly promoter-like, However, the sequence of this region di ffers from the human sequence in terms of the number, position, and ty pe of putative regulatory elements, particularly in regard to the numb er of SP-1 binding sites and the position of multiple transcription st art sites as determined by enzymatic primer extension.